Proteases, including proteinases peptidases and proteolytic enzymes, are enzymes that catalyze the cleavage of peptide bonds. The process is called proteolytic cleavage, a common mechanism of activation or inactivation of enzymes especially involved in blood coagulation or digestion. Proteases use a molecule of water for this catalysis and are thus classified as hydrolases. For example, peptidases can break either specific peptide bonds (limited proteolysis), depending on the amino acid sequence of a protein, or break down a complete peptide to amino acids (unlimited proteolysis). The activity can be a destructive change abolishing a protein's function or digesting it into its principal components, which can activate or be a signal in a signalling pathway.
Proteases have important roles in many physiological regulations including blood coagulation, fibrinolysis, complement cascading, hormone maturation, protein degradation, and apoptosis. These enzymes are involved in a multitude of physiological reactions from simple digestion of food proteins to highly regulated cascades (e.g. the blood clotting cascade, the complement system, apoptosis pathways, and the invertebrate prophenoloxidase activating cascade). Importantly, malfunctions of proteases are often involved with or are indications of diseases.
To detect and to characterize photolytic activity, the prior art discloses methods using FRET and green fluorescent proteins (GFPs) to detect proteolysis and denaturation. More particularly, such examples include monitoring protease activity by altering the FRET between a specific GFP and another fluorescent protein, such as yellow fluorescent proteins. These protease sensors have limitations for accurate quantitative measurement of enzymatic activity especially for in vivo application, which is partly attributable to problems associated with photon bleaching/activation and small signal change as well as problems for the orientation change in the living cells
Accordingly, there is a need for improved protease sensors and methods for measuring and detecting protease activity. Such protease sensors can be applied both in vivo and in vitro systems. Such protease sensors should be able to detect protease activity in microenvironments so as to be useful as probes of cellular events involving changes in such microenvironments in real time. It is to these needs among others that the present invention is directed.